One of the greatest challenges in neuroscience is to decipher the logic of the neural circuitry and link it to learning, memory, and behavior. Neural circuitry is a dynamic network that incorporates neuronal activity at a variety of spatial and temporal scales. Therefore, analysis of neural circuitry demands broad and dense sampling of neuronal activity across time and brain structures. Recent breakthroughs in modern microscope and protein based fluorescence sensors have brought this goal within reach. For example, application of genetically encoded calcium indicators, such as GCaMP3, combined with two-photon microscopy, has facilitated the large- scale recording of neural activity in a genetically-identified population at multiple time scales in awake, behaving animals. These applications have greatly advanced our understanding of the dynamics of neural circuitry and its control of behavior-a critical first step toward understanding complex brain function. Building upon the momentum of calcium imaging, the immediate need to accelerate future analyses of the dynamics of neural circuitry is to develop a broader suite of optical sensors to expand the kinds of neuronal activity that can be measured. One particular area of interest is synaptic transmission, a critical event of information processing in the brain that is difficult to access wth the optical tools currently available. There are two key questions that need to be addressed before we can develop a dynamic picture of synaptic transmission. First, we must understand how synaptic connectivity is linked to its activity; second, we must determine how different types of neurotransmitters balance with each other in a defined circuitry. Therefore, I plan to develop two classes of novel protein-based fluorescent sensors, using methods that have emerged only recently, to enable monitoring of synaptic transmission from these two different angles. For the first project outlined in this proposal, I will develop sensors specially designed for simultaneous recording of both synaptic activity and connectivity. Recently, I have been involved in developing a genetically-encoded neurotransmitter sensor (iGluSnfr) to directly measure released glutamate. This sensor, for the first time, offers the potential for monitoring excitatory synaptic activity in time and space. However, its ability to report synaptic connectivity, a piece f important information stored in the neural circuitry, is currently lacking. Therefore, I will develp strategies to split iGluSnfr into pre- and post-synaptic components. This designer sensor will permit simultaneous recording of both synaptic activity and connectivity, thus providing a way to find the synapses that are activity-dependent in a defined circuitry. For the second project outlined in this proposal, I will develop a new sensor to direct monitor inhibitory communication between neurons at synapses. It is known that based on the kind of neurotransmitters released, the communication between neurons can be either excitatory or inhibitory. Imbalanced excitatory and inhibitory synapses in specific neural circuitry have been implicated in an array of neurological disorders, including depression, addiction, autism, schizophrenia and epilepsy. Yet, optical sensors for directly monitoring inhibitory signals with needed spatiotemporal resolution are still missing. I will leverage computational modeling to redesign iGluSnFr to sense inhibitory neurotransmitters, such as ?-aminobutyric acid (GABA). Similarly, the splitting strategy to be developed in project one will be further utilized to split the GABA sensor into pre- and post-synaptic components. Taken together, a successful outcome of the proposed research would provide much needed imaging tools to enable neuroscientists to obtain a comprehensive view of both excitatory and inhibitory synapses in action at the cellular, tissue, and whole-animal level.